Molecular Diagnostics Chapter 2

Molecular Diagnostics: Fundamentals, Methods, and Clinical Applications

Gene Expression

The process of transcribing and translating DNA to utilize the information stored within

What is mRNA

Messenger RNA

Carries the information from DNA to the ribosome for translation

Transcription

Turning DNA into mRNA

RNA Polymerges II

Occurs mostly in interphase

RNA Polymerase Types

Prokaryote:

Only one type

Eukaryote:

I (rRNA in the nucleolus)

II (mRNA in the nucleus)

III (tRNA in the cytoplasm)

Phenotype Changes

Can arise from structural or chemical sequence changes in DNA

Selective Expression

Phenotype changes depending on the expression levels of certain genes

Promoter

The start site in DNA for transcription.

Relation of Transcription Initiation Site to Oris

WAY more RNA initiation sites than replication sites

RNA Polymerase Supporting Proteins

Prokaryotes: a basal transcription complex comprised of the small and large ribosomal subunits and additional sigma factors.

Eukaryotes: RNA polymerase and 20+ factors for accurate initiation

Antisense Strand

The strand of DNA used as a template for RNA.

For transcription, mRNA is complimentary to this strand.

Sense Strand

The strand of DNA NOT used as a template for RNA.

When transcribing mRNA, the RNA sequence is identical to this strand but utilizes U instead of T.

DNA/RNA Polymerase Speeds

DNA Pol: 1000 bp/s

RNA Pol: 50-100 bp/s

In which direction is RNA synthesized?

Antisense is read 3’ to 5’. RNA is made 5’ to 3’.

Does RNA synthesis require a primer to start? How?

No

The first base retains all phosphates. Subsequent bases retain only their alpha phosphate.

7-methylguanosine

A methylated guanine residue that is used to cap the 5’ end of pre-mRNA. This cap protects the end of the growing pre-mRNA and facilitates ribosomal binding/translation

Uses a 5’ to 5’ covalent bond.

mRNA Capping Steps

1. Hydrolysis of the 5’ phosphate from the pre-mRNA

2. Transfer of a guanine monophosphate to the 5’ disphosphate end

3. Methylation of the attached guanine in the N7 position.

Transcription Termination (Prokaryotes)

Protein-dependent - High levels of target protein induce termination of its own synthesis. Sometimes uses transcription factor “rho”.

Rho

A Helicase enzyme that terminates transcription by associating with RNA Pol and binding to G:C rich regions.

Rho-Induced Termination

Occurs at G:C rich regions, followed by A:T rich regions.

G:C regions are transcribed into a short double-stranded RNA hairpin which slows the elongation complex. The complex then disassociates once it reaches the A:T regions

Transcription Termination (Eukaryotes)

RNA Pol II encounters a Polyadenylation Signal (polyA site). mRNA is released from Pol II by endonuclease on the carboxyl end of Pol II.

mRNA synthesized after the polyA site is degreased by another exonuclease. Once the exonuclease catches Pol II, transcription terminates.

Polyadenylate Polymerase

Adds 20 to 200 A nucleotides to the 3’ end of new RNA transcripts.

Isoform

Different transcripts that are produced from the same gene. Due to termination at different polyA sites on the gene.

RNA Pol I Transcription Termination

Terminates at a site prior to the Sal box with the help of TTF1, a termination factor.

RNA Pol III Transcription Termination

Terminates by a run of Adenines in the temple, and requires a termination factor.

Constitutive Gene Expression

When a gene is expressed (transcribed and translated) continuously due to being in continual use.

Cis Factors

DNA sequences that mark places involved in the initiation and control of RNA synthesis.

Trans Factors

Proteins that bind to cis factors and direct the assembly of transcription complexes at the proper gene.

Operon

A series of structural genes transcribed in a single mRNA, and are separated into different individual proteins.

Induction

AKA Enzyme Adaptation

The presence of certain substrates can change gene expression. For example, growing bacteria on lactose increases all lac operon expression.

lac operon

Lactose operon in bacteria - activates when lactose is present.

lacZ: Beta-galactosidase, turns lactose into glucose and galactose.

lacY: lactose permease, transports lactose into the cell.

lacA: thiogalactoside transacetylase, transacetylates galactosides.

lacI: protein represser, binds to lacO cis factor 5’ of the lacP site

lacP: where RNA Pol binds

lac Operon Repression

When lactose is absent, a represser protein binds to the operator sequence 5’ of the operon, preventing transcription by blocking RNA Pol binding.

When lactose is present, lactose binds to the represser protein, lowering its binding affinity for the lac operon inhibition site.

Enzyme Repression

When a corepressor must bind to a repressor in order to turn off transcription.

Enzyme/other factor is required to repress.

Enzyme Induction

When an inducer binds to a repressor to inhibit the repressor and start transcription.

Enzyme/other factor is required to initiate.

mal Operon Initiation

Transcription requires an activator to bind directly to RNA Pol to turn on transcription.

Attenuation

Stems and loops in the DNA are formed by hydrogen bonding complementary bases to either allow or prevent transcription.

Transcription is affected because the RNA Pol binding site will either be exposed or sequestered.

Distal Elements

Elements thousands of base pairs away from the gene they influence.

Proximal Elements

Elements and regulatory sequences that are in close proximity to the gene they influence.

What are two examples of distal elements?

Silencers - bind and repress at distal sites

Enhancers - bind and promote at distal sites

Circular RNA

Useful by products of RNA splicing for gene expression.

Roles include protein binding, activation of transcription, and sub cellular localization.

Implicated in tissue development, stress response, and cancer.

Not subject to exonuclease due to no blunt ends.

Epigenetics

A developmental phenomenon that allows cells to take on different phenotypes without changes in their genetic material.

Chromatin

The complex of DNA and its associated proteins

Nucleosome

~150 base pairs of DNA wrapped around a complex of eight histone proteins (2 each of 2A, 2B, 3, and 4).

Histone Modifications

Affects the activity of chromatin-associated proteins and transcription factors - affects gene expression

Examples include methylation, acetylation, phosphorylation, and ubiquitination.

Histone Methylation

Attracts enzymes that further methylate the DNA. Decrease of gene expression.

Histone Acetylation

Lowers the positive charge of histones, decreasing binding strength to the DNA. Allows the DNA to be more available for DNA/RNA Pol. Increase of gene expression.

CpG Islands

C:G rich areas of DNA. Often the target for methylation of DNA. Usually found at the 3’ end of genes.

Over 45,000 in the human genome.

DNA Methylation

Occurs at CpG Islands to either inhibit or promote gene expression. The main mechanism of Imprinting.

Imprinting

The stage-specific (like stage of growth/development) or gamete-specific silencing of genes.

Stage: certain genes are on at certain times of development and off at others. Controlled by methylation.

Gamete: some genes from a specific parent are silenced, but not from the other parent

Angelman Syndrome and Prader-Willi Syndrome

Distinct conditions that are both caused by the same genetic defect on chromosome 15. The difference is determined by whether the paternal or maternal chromosome the offspring inherited had the genetic lesion.

What 4 enzymes methylate DNA?

DNMT1, DNMT3a, DNMT3b, DNMT3L

DNMT = DNA Methyltransferase

Hemimethylated DNA

When the original parent strand is methylated, and the newly synthesized strand is not.

DNMT1

Maintenance enzyme that mainly methylates hemimethylated DNA.

DNMT3

Methylates un-methylated DNA.

DNMT3L

No methylase activity due to no catalytic site. Appears to regulate DNMT3A/B activities by occupying their protein or DNA-binding sites.

Hypomethylation

When too few methyl groups are added to DNA. Cause of some cellular instabilities and possibly cancer.

Hypermethylmation

Too many methyl groups added to DNA. Can cause disease and cancer.

Ten Eleven Translocation (TET) Enzymes

Catalyze the demethylation of DNA.

RNA Methylation

Affects stability, splicing, and translation.

Can methylate: mRNA, tRNA, rRNA.

Writers

A factor that modifies histones and/or nucleic acids by adding something.

Methyltransferase, phosphorylase, acetylase.

Erasers

A factor that modifies histones and/or nucleic acids by removing something.

Deacetylase, TET enzymes, and enzymes that reverse these modifications.

Chromatin Modifiers

Writers and Erasers.

Change or maintain chromatin structure.

Modulators

Readers and Transcription Factors.

Bring about the effects of the chromatin state.

Noncoding RNA

RNA that are not translated into protein but still carry out a function, usually with gene regulation.

Piwi RNA (piRNA)

Affects transposing transcription in germ cells.

Types of Noncoding RNA

Small Noncoding RNAs (sncRNAs)

Micro RNAs (miRNAs)

Short Interfering RNAs (siRNAs)

Long Noncoding RNAs (lncRNAs)

MicroRNAs (miRNAs)

17 to 27 nt in length, derived from endogenous hairpin structures.

Control gene expression, function in cell development, and defense.

Usually only present in virally infected cells or after the introduction of foreign nucleic acids (transformation).

Small Interfering RNAs (siRNAs)

Intermediates of RNA interference (RNAi), a defense for viral invasion. 21 to 22 nucleotide dsRNAs.

Used to regulate gene expression - turn off expression in lab settings.

Ribonuclease III

Enzyme responsible for generating siRNA, miRNA, and other smaller RNA from a dsRNA precursor.

miRNA Gene Regulation

miRNA can regulate gene expression by binding to the 3’ end of mRNA, preventing translation.

RNase III

Endonuclease, Dicer, Drosha

Cleave pre-miRNA into miRNA hairpins

RNA-Induced Silencing Complex (RISC)

A multiprotein complex that incorporates short RNA molecules, typically siRNA or miRNA, guiding the complex to complementary mRNA targets for degradation or translational repression.

RNA Interference (RNAi)

Small RNA, such as siRNA, can inhibit gene expression and/or translation of target RNA molecules.

Long Noncoding RNAs (lncRNAs)

Regulate chromatin structure which affects gene expression and disease processes.

Xist

A special lncRNA that is responsible for X-chromosome inactivation in female cells. Expressed from the X Inactivation Center (XIC) on chromosome X.

Forms a complex with other XIC proteins that methylates the X chromosome, silencing all genes on one X chromosome.

Tsix

Complementary lncRNA to Xist - negatively regulates Xist as an anti-sense RNA.